Recombinant HIV-1 vaccine candidates based on replication-defective flavivirus vector.

Multiple approaches utilizing viral and DNA vectors have shown promise in the development of an effective vaccine against HIV. In this study, an alternative replication-defective flavivirus vector, RepliVax (RV), was evaluated for the delivery of HIV-1 immunogens. Recombinant RV-HIV viruses were engineered to stably express clade C virus Gag and Env (gp120TM) proteins and propagated in Vero helper cells. RV-based vectors enabled efficient expression and correct maturation of Gag and gp120TM proteins, were apathogenic in a sensitive suckling mouse neurovirulence test, and were similar in immunogenicity to recombinant poxvirus NYVAC-HIV vectors in homologous or heterologous prime-boost combinations in mice. In a pilot NHP study, immunogenicity of RV-HIV viruses used as a prime or boost for DNA or NYVAC candidates was compared to a DNA prime/NYVAC boost benchmark scheme when administered together with adjuvanted gp120 protein. Similar neutralizing antibody titers, binding IgG titers measured against a broad panel of Env and Gag antigens, and ADCC responses were observed in the groups throughout the course of the study, and T cell responses were elicited. The entire data demonstrate that RV vectors have the potential as novel HIV-1 vaccine components for use in combination with other promising candidates to develop new effective vaccination strategies.

Construction, safety, and immunogenicity in nonhuman primates of a chimeric yellow fever-dengue virus tetravalent vaccine.

We previously reported construction of a chimeric yellow fever-dengue type 2 virus (YF/DEN2) and determined its safety and protective efficacy in rhesus monkeys (F. Guirakhoo et al., J. Virol. 74:5477-5485, 2000). In this paper, we describe construction of three additional YF/DEN chimeras using premembrane (prM) and envelope (E) genes of wild-type (WT) clinical isolates: DEN1 (strain PUO359, isolated in 1980 in Thailand), DEN3 (strain PaH881/88, isolated in 1988 in Thailand), and DEN4 (strain 1228, isolated in 1978 in Indonesia). These chimeric viruses (YF/DEN1, YF/DEN3, and YF/DEN4) replicated to ~7.5 log(10) PFU/ml in Vero cells, were not neurovirulent in 3- to 4-week-old ICR mice inoculated by the intracerebral route, and were immunogenic in monkeys. All rhesus monkeys inoculated subcutaneously with one dose of these chimeric viruses (as monovalent or tetravalent formulation) developed viremia with magnitudes similar to that of the YF 17D vaccine strain (YF-VAX) but significantly lower than those of their parent WT viruses. Eight of nine monkeys inoculated with monovalent YF/DEN1 -3, or -4 vaccine and six of six monkeys inoculated with tetravalent YF/DEN1-4 vaccine seroconverted after a single dose. When monkeys were boosted with a tetravalent YF/DEN1-4 dose 6 months later, four of nine monkeys in the monovalent YF/DEN groups developed low levels of viremia, whereas no viremia was detected in any animals previously inoculated with either YF/DEN1-4 vaccine or WT DEN virus. An anamnestic response was observed in all monkeys after the second dose. No statistically significant difference in levels of neutralizing antibodies was observed between YF virus-immune and nonimmune monkeys which received the tetravalent YF/DEN1-4 vaccine or between tetravalent YF/DEN1-4-immune and nonimmune monkeys which received the YF-VAX. However, preimmune monkeys developed either no detectable viremia or a level of viremia lower than that in nonimmune controls. This is the first recombinant tetravalent dengue vaccine successfully evaluated in nonhuman primates.

Phenotypic and molecular analyses of yellow fever 17DD vaccine viruses associated with serious adverse events in Brazil.

The yellow fever (YF) 17D virus is one of the most successful vaccines developed to data. Its use has been estimated to be over 400 million doses with an excellent record of safety. In the past 3 years, yellow fever vaccination was intensified in Brazil in response to higher risk of urban outbreaks of the disease. Two fatal adverse events temporally associated with YF vaccination were reported. Both cases had features similar to yellow fever disease, including hepatitis and multiorgan failure. Two different lots of YF 17DD virus vaccine were administered to the affected patients and also to hundreds of thousands of other individuals without any other reported serious adverse events. The lots were prepared from the secondary seed, which has been in continuous use since 1984. Nucleotide sequencing revealed minor variations at some nucleotide positions between the secondary seed lot virus and the virus isolates from patients; these differences were not consistent across the isolates, represented differences in the relative amount of each nucleotide in a heterogeneous position, and did not result in amino acid substitutions. Inoculation of rhesus monkeys with the viruses isolated from the two patients by the intracerebral (ic) or intrahepatic (ih) route caused minimal viremia and no clinical signs of infection or alterations in laboratory markers. Central nervous system histological scores of rhesus monkeys inoculated ic were within the expected range, and there were no histopathological lesions in animals inoculated ih. Altogether, these results demonstrated the genetic stability and attenuated phenotype of the viruses that caused fatal illness in the two patients. Therefore, the fatal adverse events experienced by the vaccinees are related to individual, genetically determined host factors that regulate cellular susceptibility to yellow fever virus. Such increased susceptibility, resulting in clinically overt disease expression, appears to be extremely rare.

Development of a rubella virus vaccine expression vector: use of a picornavirus internal ribosome entry site increases stability of expression.

Rubella virus (RUB) is a small plus-strand RNA virus classified in the Rubivirus genus of the family Togaviridae. Live, attenuated RUB vaccines have been successfully used in vaccination programs for over 25 years, making RUB an attractive vaccine vector. In this study, such a vector was constructed using a recently developed RUB infectious cDNA clone (Robo). Using a standard strategy employed to produce expression and vaccine vectors with other togaviruses, the subgenomic promoter was duplicated to produce a recombinant construct (termed dsRobo) that expressed reporter genes such as chloramphenicol acetyltransferase and green fluorescent protein (GFP) under control of the second subgenomic promoter. However, expression of the reporter genes, as exemplified by GFP expression by dsRobo/GFP virus, was unstable during passaging, apparently due to homologous recombination between the subgenomic promoters leading to deletion of the GFP gene. To improve the stability of the vector, the internal ribosome entry site (IRES) of a picornavirus, encephalomyocarditis virus, was used instead of the second subgenomic promoter to eliminate homology. Construction was initiated by first replacing the subgenomic promoter in the parent Robo infectious clone with the IRES. Surprisingly, viable virus resulted; this virus did not synthesize a subgenomic RNA. The subgenomic promoter was then reintroduced in an orientation such that a single subgenomic RNA was produced, GFP was the initial gene on this RNA, while the RUB structural protein open reading frame was downstream and under control of the IRES element. GFP expression by this vector was significantly improved in comparison to dsRobo/GFP. This strategy should be applicable to increase the stability of other togavirus vectors.

Infectious cDNA clone of the RA27/3 vaccine strain of Rubella virus.

Rubella virus (RUB), a small plus-strand RNA virus, is a significant human pathogen. The RA27/3 vaccine strain of RUB is one of the most successful live attenuated vaccines developed. In this article, we report the construction of an RA27/3 infectious clone, a complete cDNA copy of the RA27/3 genome that can be transcribed in vitro to generate infectious RNA molecules. Virus generated from such in vitro transcripts was phenotypically similar to RA27/3 virus. To investigate the attenuation of the RA27/3 strain, a series of chimeras was made by the insertion of different fragments of the RA27/3 genome into an infectious clone based on the Therien wild-type strain of RUB. Analysis of the resulting chimeric viruses revealed that the pattern of RA27/3 attenuation in cell culture is complex: attenuating elements in the RA27/3 genome were found in the 5' untranslated region (UTR), a region of the nonstructural proteins containing the protease motif and the capsid gene. Within the 5' UTR, the attenuation determinant was mapped to nt 7. Surprisingly, these analyses also revealed a potentiating mutation at nt 164 of the RA27/3 genome. Although this determinant was within the coding sequences of the nonstructural proteins, the encoded amino acid had no effect on cell culture phenotype and thus the determinant may operate at the level of RNA structure. In addition to investigation of the mechanisms of RA27/3 attenuation, the availability of the RA27/3 infectious clone offers the opportunity for strict genetic control over RUB vaccine manufacturing, for development of novel DNA-based vaccines against RUB, and for development of recombinant RUB vaccines that also target other diseases.

Rubella virus induces apoptosis in culture cells.

The replication of rubella virus (RUB) in Vero cells, an adherent cell line, results in apoptotic death of infected cells as detected by chromatin fragmentation assays. In infected cultures, virtually all of the cells that had become detached (a hallmark feature of RUB-induced cytopathology) were apoptotic; they were predominantly dead as shown by propidium iodide and trypan blue exclusion tests. In contrast, the majority of the cells in the infected monolayers that remained adherent were alive and contained intact chromatin. Thus simple counting of detached cells in the medium is a convenient way of measuring the extent of RUB-induced apoptosis. RUB-induced cytopathology was inhibited by z-VAD-fmk, an inhibitor of caspases that are involved in the execution stages of apoptosis, confirming the induction of apoptosis by RUB. The lack of apoptotic adherent cells (maximally 1% at any time point through 6 days postinfection) indicates that the induction of apoptosis is asynchronous since cells become uniformly virus antigen-positive by day 2 postinfection. To elucidate whether this asynchronicity and the ability of RUB to persistently infect Vero cells were due to a suppression of apoptosis, we examined whether RUB can suppress chemically induced apoptosis. Staurosporine (ST) was found to be an efficient inducer of apoptosis in Vero cells. ST treatment of RUB-infected and RUB persistently infected cells resulted in a much higher proportion of detached cells, higher even than in Vero cells treated with ST alone. This indicates that RUB does not suppress ST-induced apoptosis and, rather, that ST and RUB acted cumulatively in inducing apoptosis, possibly indicating that they use different induction pathways.

Effects of defined mutations in the 5' nontranslated region of rubella virus genomic RNA on virus viability and macromolecule synthesis.

The 5' end of the genomic RNA of rubella virus (RUB) contains a 14-nucleotide (nt) single-stranded leader (ss-leader) followed by a stem-and-loop structure [5'(+)SL] (nt 15 to 65), the complement of which at the 3' end of the minus-strand RNA [3'(-)SL] has been proposed to function as a promoter for synthesis of genomic plus strands. A second intriguing feature of the 5' end of the RUB genomic RNA is the presence of a short (17 codons) open reading frame (ORF) located between nt 3 and 54; the ORF encoding the viral nonstructural proteins (NSPs) initiates at nt 41 in an alternate translational frame. To address the functional significance of these features, we compared the 5'-terminal sequences of six different strains of RUB, with the result that the short ORF is preserved (although the coding sequence is not conserved) as is the stem part of both the 5'(+)SL and 3'(-)SL, while the upper loop part of both structures varies. Next, using Robo302, an infectious cDNA clone of RUB, we introduced 31 different mutations into the 5'-terminal noncoding region, and their effects on virus replication and macromolecular synthesis were examined. This mutagenesis revealed that the short ORF is not essential for virus replication. The AA dinucleotide at nt 2 and 3 is of critical importance since point mutations and deletions that altered or removed both of these nucleotides were lethal. None of the other mutations within either the ss-leader or the 5'(+)SL [and accordingly within the 3'(-)SL], including deletions of up to 15 nt from the 5'(+)SL and three different multiple-point mutations that lead to destabilization of the 5'(+)SL, were lethal. Some of the mutations within both ss-leader and the 5'(+)SL resulted in viruses that grew to lower titers than the wild-type virus and formed opaque and/or small plaques; in general mutations within the stem had a more profound effect on viral phenotype than did mutations in either the ss-leader or upper loop. Mutations in the 5'(+)SL, but not in the ss-leader, resulted in a significant reduction in NSP synthesis, indicating that this structure is important for efficient translation of the NSP ORF. In contrast, viral plus-strand RNA synthesis was unaffected by the 5'(+)SL mutations as well as the ss-leader mutations, which argues against the proposed function of the 3'(-)SL as a promoter for initiation of the genomic plus-strand RNA.

Genomic sequence of the RA27/3 vaccine strain of rubella virus.

The sequence of the genome of the RA27/3 vaccine strain of rubella virus (RUB) was determined. In the process, several discrepancies between the previously reported genomic sequences of two wild RUB strains (Therien and M33) were resolved. The genomes of all three strains contain 9762 nucleotides (nts), exclusive of the 3' poly A tract. In all three strains, the genome contains (5' to 3'), a 40 nt 5' untranslated region (UTR), an open reading frame (ORF) of 6348 nts that encodes nonstructural proteins, a 123 nt UTR between the two genomic ORFs, a 3189 nt ORF that encodes the structural proteins, and a 62 nt 3' UTR. The 5' end of the subgenomic RNA was found to correspond to a uridine residue at nt 6436 of the genomic RNA. At the nucleotide level, the sequence of the three strains varied by 1.0 to 2.8%, while at the amino acid level, the sequence varied by 1.1 to 2.4% over both ORFs. The RA27/3 sequence will be of use in identification of the determinants of its attenuation, in vaccine production control and in development of second generation RUB vaccines based on recombinant DNA technology.

Improvement of the specific infectivity of the rubella virus (RUB) infectious clone: determinants of cytopathogenicity induced by RUB map to the nonstructural proteins.

A plasmid, Robo102, which contains a cDNA copy of the rubella virus (RUB) genomic RNA from which infectious transcripts can be synthesized in vitro, was recently developed (C. Y. Wang, G. Dominguez, and T. K. Frey, J. Virol. 68:3550-3557, 1994). To increase the specific infectivity of Robo102 transcripts (approximately 5 plaques/10 microg of transcripts), a modified reverse transcription-PCR method was used to amplify nearly 90% of the RUB genome in three fragments, which were then used to replace the corresponding fragments in Robo102. Replacement of a fragment covering nucleotides (nt) 5352 to 9759 of the RUB genome yielded a construct, Robo202, which produced highly infectious transcripts (10(4) plaques/microg), indicating the presence of an unrecognized deleterious mutation (or mutations) in this region of the Robo102 cDNA. Robo102 was based on the w-Therien strain of RUB, which forms opaque plaques in Vero cells, while the PCR replacement fragments were generated from a variant, f-Therien, which produces clear plaques in Vero cells. Although Robo202 contains over 4,000 nt from f-Therien, Robo202 virus produces opaque plaques. However, when the other two PCR fragments amplified from f-Therien (nt 1 to 1723 and nt 2800 to 5352) were introduced into Robo202, the resulting construct, Robo302, yielded transcripts that produced a virus that formed clear plaques. This indicates that the determinants of plaque morphology map to the regions of the genome covered by these two fragments, both of which are in the nonstructural open reading frame. Generation of Robo202/302 chimeras indicated that the most 5' terminal fragment (nt. 1 to 1723) had the greatest effect on plaque morphology. The plaque morphology was correlated with the ability of the viruses to kill infected cells. The only difference at the molecular level detected among the viruses was that the more cytopathic viruses produced more nonstructural proteins than did the less cytopathic viruses. This finding, as well as the mapping of the genetic determinants to the region of the genome encoding these proteins, indicates that the nonstructural proteins can mediate cell killing.

Double-subgenomic Sindbis virus recombinants expressing immunogenic proteins of Japanese encephalitis virus induce significant protection in mice against lethal JEV infection.

A series of double-subgenomic Sindbis virus (dsSIN) recombinants that express cassettes encoding the immunogenic proteins of Japanese encephalitis virus (JEV) [prM-E, prM-E-NS1, NS1-NS2A, 80%E (encodes the amino-terminal 80% part of E), and NS1] were constructed and analyzed for their ability to confer protective immunity in mice against lethal challenge with neurovirulent JEV. The cassettes were introduced into both 5' [second subgenomic promoter of the vector precedes the SIN structural open reading frame (SP-ORF)] and 3' (the promoter follows the SP-ORF) dsSIN vectors. The longest cassette (prM-E-NS1) was 3.2 kb in length, which is remarkable for such a small vector virus as SIN (SIN genome is roughly 11.8 kb in length). The level of expression of JEV proteins appeared similar for both 5' and 3' recombinants. In general, the stability of the recombinants obtained was found to be low (expression was lost following one to five passages at low multiplicity of infection, depending on the recombinant). However, 5' recombinants containing longer cassettes (prM-E-NS1, prM-E, NS1-NS2A) were more stable than the corresponding 3' recombinants. Intraperitoneal inoculation of mice with 10(7) PFU of dsSIN-JEV recombinants induced antibodies against JEV proteins and low titers of JEV-neutralizing antibodies were produced by mice inoculated with recombinants expressing 80%E, prM-E, and prM-E-NS1. A single immunization of mice with the dsSIN-prM-E or dsSIN-prM-E-NS1 recombinants provided 40-65% protection against peripheral lethal challenge with 10(4) LD50 of neurovirulent JEV. The results demonstrate that genetically engineered togaviruses can be successfully used as vaccine vectors.

Sindbis vectors suppress secretion of subviral particles of Japanese encephalitis virus from mammalian cells infected with SIN-JEV recombinants.

Double-subgenomic Sindbis virus (dsSIN) recombinants that express cassettes encoding prM-E or a C-terminally truncated form of E of Japanese encephalitis virus (JEV) were constructed. The products were efficiently expressed in both mammalian and mosquito cell lines infected with the dsSIN recombinants. However, suppression of prM-E secretion from mammalian cells infected with dsSIN-prM-E recombinants was observed. This suppression was more pronounced late in infection (< 5% of total product was secreted during an 8-hr chase) than early in infection (15% secretion during a 6-hr chase). In comparison, a vaccinia virus-prM-E recombinant (vP829) described previously (E. Konishi et al. (1991) Virology 185, 401-410) was shown to secrete 35-50% of total product during a 6- to 8-hr chase both early and late in infection. In contrast, secretion of prM-E from dsSIN-prM-E-infected mosquito (C6/36) cells was found to be efficient (> 50% during an 8-hr chase). The prM-E secreted from both mammalian and mosquito cells was in the form of subviral particles as determined by velocity gradient centrifugation, sensitivity to nonionic detergent, and analysis of processing of N-linked glycans. The truncated E protein expressed by the dsSIN recombinants was secreted efficiently from both mammalian and mosquito cells. Coinfection experiments with the dsSIN-JEV recombinants + wild-type vaccinia virus and vP829 + SIN demonstrated that the reduced level of secretion of subviral particles exhibited by the dsSIN-JEV recombinants was due to an inhibitory effect of the dsSIN vectors. Furthermore, this inhibitory effect was accounted for by the SIN nonstructural proteins since SIN replicons that express prM-E cassette in place of the SIN structural protein open reading frame exhibited a low level of subviral particle secretion. No self-propagating infectious particles were produced in cells transfected with SIN replicons that encode the JEV prM-E cassette. The suppression of subviral particle secretion was apparently correlated with the inhibition of cell protein synthesis which is mediated in SIN-infected vertebrate cells by expression of the SIN nonstructural proteins.

Comparative analysis of serological activity of non-structural protein (NS1) from tick-borne encephalitis virus and its analog expressed in bacterial cells.

By means of immunoaffinity chromatography and expression of the gene in Escherichia coli, non-structural glycoprotein NS1 of tick-borne encephalitis virus (TBEV) and its recombinant analog were prepared. Antisera against these proteins were obtained by hyperimmunisation of rabbits. The antisera were tested by means of complement fixation, agar diffusion, hemagglutination inhibition and virus neutralization. Although both antisera are reacted with natural antigen, the recombinant analog of NS1 did not bind antibodies against natural protein in complement fixation and immunoprecipitation. Nevertheless the NS1 analog was rather active in ELISA. Neither the natural nor the recombinant protein protected experimental animals from lethal virus infection. A contamination of natural NS1 antigen with small amounts of structural glycoprotein E may be responsible for both antibody formation and virus neutralization. This can be relevant for the design of a subunit vaccine.

Site-directed mutagenesis of the tick-borne encephalitis virus NS3 gene reveals the putative serine protease domain of the NS3 protein.

Several mutations were introduced into the putative serine protease domain of the tick-borne encephalitis virus NS3 protein and into a possible internal cleavage site within the protein. The influence of these mutations on proteolytic activity of NS3 protein and NS3' protein formation was tested in vitro. It was found that NS3' formation was not dependent on the activity of the NS3 N-terminal serine protease. Mutations affecting the Ser-138 residue of the NS3 protein prohibited cleavage between NS2B and NS3 proteins when the NS2B-NS3 part of the viral genome was expressed in vitro, suggesting the key role of Ser-138 in viral serine protease functioning.

A short form of the tick-borne encephalitis virus NS3 protein.

Using monoclonal antibodies to the tick-borne encephalitis virus (TBE) nonstructural protein NS3 two forms of this protein were revealed in TBE-infected mammalian cells: a full-length form (69 kDa) and a short form (49 kDa) which has not been observed before and was called NS3'. Recombinant plasmids were constructed and various fragments of the TBE NS3 gene were expressed in rabbit reticulocyte lysate. By analyzing immune precipitates of 35S-labeled translation products, we could monitor and localize internal cleavage of NS3, due to which the NS3' protein was generated.

[Expression of the gene for tick-borne viral encephalitis virus NS3 protein in Escherichia coli cells]. / Ekspressiia v kletkakh Escherichia coli gena belka NS3 virusa kleshchevogo éntsefalita.

On the base of two overlapping cDNA-clones of tick-borne encephalitis virus (TBEV) genome and synthetic DNA fragments full DNA-copy of the TBEV NS3 protein gene was constructed and expressed in the E. coli cells. It was demonstrated that the relatively low biosynthesis level of full-length NS3 protein in the bacteria was due to the toxicity of the N-terminal region of the protein, consisting of it's first 180 amino acid residues. A form of the gene with deletion of nucleotides coding for the toxic region (called NS3*) was constructed and effective bacterial product of NS3* protein was obtained. The panel of monoclonal antibodies to TBEV NS1 and NS3 proteins was generated. According to the results of experiments of the binding of the monoclonal antibodies 18B2 to the bacterial products of NS3 and NS3* genes it was concluded, that the antigenic determinant recognized by these antibodies was located between 174 and 236 amino acids of TBEV NS3 protein.

[Expression of gene coding for the NS1 protein of the tick-borne encephalitis virus in Escherichia coli cell]. / Ekspressiia v kletkakh Escherichia coli gena belka NS1 virusa kleshchevogo éntsefalita.

Two possible forms of tick-borne encephalitis virus (TBE) gene NS1 (called NS1' and NS1) were constructed using two overlapping cDNA-fragments of TBE genome and synthetic DNA fragments. This genes were expressed in E. coli cells in expression vector pUR290 as individual proteins or fusion with bacterial beta-galactosidase. The proteins NS1 (Mw. 39 kDa), beta-galactosidase-NS1' (Mw. 162 kDa) and beta-galactosidase-NS1 (Mw. 155 kDa) were effectively synthesized under the Plgc-promoter induction conditions. Expression of NS1' gene results in the formation of two virus-specific proteins (Mw. 46 and 44 kDa). All bacterial analogs of NS1 protein fixed monoclonal and polyclonal antibodies specific to viral NS1.